The neuromuscular junction (NMJ), a synapse between a muscle fiber and axon of a motorneuron is one of the most studied synapses in the human body. There are still many elements of synapse biology which are not known. Intracellular signals originating from the neuron, agrin and the neurotransmitter, acetylcholine (ACh) are responsible for the assembly and maturation of the synapse, but initial postsynaptic differentiation may involve both the muscle and motorneuron and not solely dictated by the nerve terminal as once proposed. The mechanism of synapse formation is still not fully understood. Techniques based on soft lithography are proposed to study spatiotemporal behavior of synapse biology. Artificial neurons are replicated in microfluidic capillaries with diameters similar to those an axon. The spatiotemporal behavior of acetylcholine receptors (AChR) clustering dynamics is studied in-vitro by local applications of agrin and ACh. There is evidence to suggest that agrin can cause cluster assembly over spatial domains much larger than the area applied, suggesting an intracellular mechanism on muscle surface may signal the active formation of a synapse. Even less is known about the spatial behavior of ACh, and its role as an AChR cluster antagonist hasn't been studied using spatially resolved techniques. Microfluidic channels capable of independently culturing neurons and myotubes will be utilized to a reconstitute a NMJ in-vitro to investigate the kinetics and dynamics of nerve innervation. As the NMJ goes through a dynamic development, the relatively flat architecture of the embryonic postsynaptic apparatus become three -dimensional forming folds upon which AChR clusters. The origin or significance of these folds is unknown. Muscle cell culture on topographic surfaces fabricated by replica molding will probe whether topographic cues can induce AChRs clustering, while the formation of folds in-vitro will be examined over a series of complaint culture substrata with mechanical properties similar to in- vivo conditions. Relevance to Public Health: This proposal examines the biology of the neuromuscular junction into a fully functioning synapse. Novel methods are employed to study the role of intercellular signaling on the proper function in order to understand, for example how damaged synapses may be repaired. [unreadable] [unreadable]